Competition of milrinone, a non-iodinated cardiac inotropic agent, with thyroid hormone for binding sites on human serum prealbumin (TBPA)

Abstract

Milrinone [2-methyl-5-cyano-(3,4'-bipyridin)-6(1 H)-one] is a positive cardiac inotropic agent recently shown to have thyromimetic activity in vitro in a rabbit myocardial membrane Ca 2+-ATPase system [K. M. Mylotte et al., Proc. natn. Acad. Sci. U.S.A. 82, 7974 (1985)]. In the present studies, milrinone was examined for activity as an inhibitor of iodothyronine binding by human serum thyroid hormone transport proteins, thyroxine-binding globulin (TBG), prealbumin (TBPA) and albumin. Polyacrylamide gel electrophoresis at pH9.0 of sera equilibrated with [ 125I]thyroxine showed that milrinone competed with l-thyroxine (T 4) for binding sites on TBPA (10 and 100 μM milrinone caused 61 and 73% reductions, respectively, in T 4 binding to TBPA, P < 0.01); T 4 displaced from TBPA was bound by TBG and albumin. Comparable reductions in T 4 binding to TBPA were observed in electrophoretic studies conducted at pH 7.4. Binding of triiodo- l-thyronine (T 3) to TBPA was electrophoretically confirmed and shown to be decreased in the presence of milrinone. Electrophoresis of purified TBPA also demonstrated that [ 14C]milrinone co-migrated with this transport protein and that milrinone displaced tracer T 4 from TBPA. Amrinone, the 2-H-5-NH 2 analog of milrinone, had less than 5% of the activity of milrinone as an inhibitor of T 4 binding in electrophoretic studies. Scatchard analysis of T 4 and milrinone binding to purified TBPA, measured by equilibrium dialysis, showed two classes of binding sites, with association constants, respectively, of 6.1 × 10 7 M −1 and 1.6 × 10 6 M −1 for T 4, and 1.7 × 10 6 M −1 and 8.9 × 10 2 M −1 for milrinone. Computer graphic modeling of the binding of milrinone to the T 4 site in the crystal structure of TBPA showed that milrinone best occupied this site when the substituted bipyridine ring overlapped the phenolic ring of T 4. In this orientation the 5-cyano group, which has an electronegativity similar to that of iodine, occupied the same volume as the 5'-iodine of T 4. The 5-amino group of amrinone lacks these characteristics. In this orientation, the keto function of milrinone overlapped the T 4 4'-hydroxyl and could participate in similar intermolecular interactions. Thus, milrinone, a non-iodinated bipyridine, and thyroid hormone share structural and biochemical homologies and compete for the same binding site on TBPA.